Tetracycline fluorescence as calcium-probe for nerve membrane with some model studies using erythrocyte ghosts

Summary The tetracycline dyes, particularly chlorotetracycline, have been employed as probes of membrane-associated calcium during the excitation process of nerve. Both squid giant axons, stained internally, and lobster nerves, stained externally, show a small increase in fluorescent light during the action potential. Increasing the calcium concentration bathing a lobster nerve leads to a larger optical signal. Adding fluoride ion to the inside of a squid axon, which might be expected to influence the internal calcium-ion concentration, also leads to a larger optical signal. Squid axons have been studied under conditions of voltage clamp and the hyperpolarizing response. Model studies were done with erythrocyte ghosts to clarify the influence of membranes and calcium on the fluores-cence of the tetracyclines. Chlorotetracycline may be monitoring calcium concentration associated with the inner surface of the nerve membrane.     

Effect of changes in temperature on the cholesterol and phospholipid ratio in nerve fibers of the frog and squid

The character of changes in molar correlation of cholesterol and phospholipids has been studied at different functional states in frog myelinic nerve and squid nonmyelinic nerve trunk. The correlation cholesterol/phospholipid (C/P) found for both nerves in rest increases at temperature 38 degrees C. The electrical stimulation on the background of higher temperature action leads to unequal shift of C/P: the increase compared with the rest in frog and decrease in squid.     

Radular mechanosensory neuron in the buccal ganglia of the terrestrial slug,Incilaria fruhstorferi

A radular mechanosensory neuron, RM, was identified in the buccal ganglia of Incilaria fruhstorferi. Fine neurites ramified bilaterally in the buccal ganglia, and main neurites entered the subradular epithelium via buccal nerve 3 (n3). When the radula was distorted by bending, RM produced an afferent spike which was preceded by an axonic spike recorded at n3. The response of RM to radular distortion was observed even in the absence of Ca²⁺, which drastically suppressed chemical synaptic interactions. Therefore, RM was concluded to be a primary radular mechanoreceptor.During rhythmic buccal motor activity induced by food or electrical stimulation of the cerebrobuccal connective, RM received excitatory input during the radular retraction phase. In the isolated buccal ganglia connected to the radula via n3s, the afferent spike, which had been evoked by electrical stimulation of the subradular epithelium, was broadened with the phasic excitatory input. Since the afferent spike was also broadened by current injection into the soma, depolarization due to the phasic input may have produced the spike broadening.Spike broadening was also observed during repetitive firing evoked by current injection. The amplitude of the excitatory postsynaptic potential in a follower neuron increased depending on the spike broadening of RM.Abbreviations CBC cerebrobuccal connective - EPSP excitatory postsynaptic potential - n1,n3 buccal nerves 1 and 3 - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

Enhancement of peripheral nerve regeneration due to treadmill training and electrical stimulation is dependent on androgen receptor signaling

Moderate exercise in the form of treadmill training and brief electrical nerve stimulation both enhance axon regeneration after peripheral nerve injury. Different regimens of exercise are required to enhance axon regeneration in male and female mice (Wood et al.: Dev Neurobiol 72 (2012) 688–698), and androgens are suspected to be involved. We treated mice with the androgen receptor blocker, flutamide, during either exercise or electrical stimulation, to evaluate the role of androgen receptor signaling in these activity‐based methods of enhancing axon regeneration. The common fibular (CF) and tibial (TIB) nerves of thy‐1‐YFP‐H mice, in which axons in peripheral nerves are marked by yellow fluorescent protein (YFP), were transected and repaired using CF and TIB nerve grafts harvested from non‐fluorescent donor mice. Silastic capsules filled with flutamide were implanted subcutaneously to release the drug continuously. Exercised mice were treadmill trained 5 days/week for 2 weeks, starting on the third day post‐transection. For electrical stimulation, the sciatic nerve was stimulated continuously for 1 h prior to nerve transection. After 2 weeks, lengths of YFP+ profiles of regenerating axons were measured from harvested nerves. Both exercise and electrical stimulation enhanced axon regeneration, but this enhancement was blocked completely by flutamide treatments. Signaling through androgen receptors is necessary for the enhancing effects of treadmill exercise or electrical stimulation on axon regeneration in cut peripheral nerves. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 531–540, 2014     

Activation of cerebral neurons by static nerve inputs in Aplysia californica

A number of cerebral B-neurons of Aplysia californica were activated by tactile stimulation of the statocysts and by electrical stimulation of the static nerve. Types of responses recorded included antidromic spike, monosynaptic EPSP with or without spike and polysynaptic EPSPs. Some of the B-neurons were inhibited by trains of electrical stimulation of the static nerve. Hyperpolarization was mostly preceded by a short increase of spiking, usually during stimulation. Some A-neurons also responded with inhibition. The statocyst nerve contained axons carrying information not only from the statocysts to the cerebral ganglion but also from the cerebral ganglion to the statocyst. The latter pathway could be activated by tactile stimulation of the tentacles. Activation of either static nerves resulted in the increase of activity of the other static nerve through the cerebral ganglion, suggesting interaction between the two statocysts.     

The influence of an unmyelinated terminal on repetitive firing of a mammalian receptor afferent fiber

The distal end of a myelinated receptor afferent fiber consists of an unmyelinated terminal membrane which is assumed to be the site of sensory transduction, whereas the action potential encoding appears at a distal node of Ranvier. In the present paper a model of a mammalian myelinated nerve fiber was augmented by an unmyelinated terminal segment into which stimulating current was injected thus modelling the situation at a myelinated receptor afferent fiber. It was found that the introduction of the unmyelinated terminal reduces the repetitive firing rate shown by the model. However, also the amplitude of the spikes at the site of action potential generation diminishes through the large electrical load which the unmyelinated terminal imposes onto the active parts of the nerve fiber model. This "loss" of spike amplitude can abolish the ability of the model to show repetitive activity, if the unmyelinated terminal increases in size. On the other hand, the incorporation of sodium channels into the terminal membrane compensates the spike amplitude reduction introduced by the electrical load of that membrane. This allows repetitive firing at a lower frequency than would be possible for a model with an equivalent sodium-channel-free terminal. The results show that the unmyelinated terminal present at the distal end of myelinated receptor afferent fibers has not only the ability to provide sensory transduction but evokes also a reduction in the discharge rate of the encoding membrane.     

Depth-resolved measurement of transient structural changes during action potential propagation

We report noncontact optical measurement of fast transient structural changes in the crustacean nerve during action potential propagation without the need for exogenous chemicals or reflection coatings. The technique, spectral domain optical coherence tomography, provides real-time cross-sectional images of the nerve with micron-scale resolution to select a specific region for functional assessment and interferometric phase sensitivity for subnanometer-scale motion detection. Noncontact optical measurements demonstrate nanometer-scale transient movement on a 1-ms timescale associated with action potential propagation in crayfish and lobster nerves.     

Mechanisms of bioelectric activity in electric tissue. I. The response to indirect and direct stimulation of electroplaques of Electrophorus electricus

1. A preparation is described consisting of one or several layers of innervated cells of the electric organ of Electrophorus electricus. 2. Each plaque is multiply innervated and only at its caudal face. The nerve fibers may derive from two or more different nerve trunks. 3. During activity the innervated face becomes negative relative to the non-innervated. 4. The first electrical response of the cell to an increasing neural volley is graded and has the character of a prepotential. At a critical size of the prepotential the cell discharges with an all-or-nothing spike. 5. Both responses have durations of about 2 msec. 6. A neural volley which does not cause the spike discharge facilitates the discharge of the cell by a second subsequent volley in the same nerve (temporal facilitation). 7. The period of facilitation lasts ca. 900 msec. During the first 100 msec., the facilitation is large enough to cause a spike. In the later portion only the prepotential is facilitated. No electrical concomitant has been detected. 8. Neural volleys reaching the plaque from different trunks interact at the cell to produce a period of facilitation lasting only about 2 msec. This interaction is interpreted as spatial summation. 9. In a population of cells, simultaneous stimulation of 2 nerves causes a smaller discharge than the sum of the two isolated responses (occlusion). 10. Cells denervated for 7 weeks or more can be excited directly, but only by a current flow outward through the caudal face. 11. Weak direct stimulation causes a prepotential in the denervated plaque. On increasing the stimulus the prepotential increases to a critical size when a spike develops. The duration of both responses is about 2 msec. 12. The absolutely refractory period of the denervated cell is about 1.5 msec. and relative refractoriness lasts about 15 msec. 13. Direct stimulation causes slight facilitation lasting as long as 200 msec. 14. Repetitive stimulation of the nerve at low frequencies (2 to 3 per second) causes rapid "fatigue" of transmission. The denervated plaque, however, responds for several minutes to repetitive direct stimulation at high frequencies (25 per second).     

Mechanisms controlling choline transport and acetylcholine synthesis in motor nerve terminals during electrical stimulation

Electrical stimulation of the chick ciliary nerve leads to a frequency-dependent increase in the Na+-dependent high affinity uptake of [3H]choline (SDHACU) and its conversion to acetylcholine (ACh) in the nerve terminals innervating the iris muscle. The forces that drive this choline (Ch) uptake across the presynaptic membrane were evaluated. Depolarization with increased [K+] out or veratridine decreases Ch accumulation. In addition to the electrical driving force, energy is provided by the Na+ gradient. Inhibition of the Na,K-ATPase decreased the Ch taken up. Thus, changes in the rate of Ch transport are dependent on the electrochemical gradients for both Ch and Na+. Ch uptake and ACh synthesis were increased after a conditioning preincubation with high [K+] out or veratridine. As is the case for electrical stimulation, this acceleration of Ch uptake and ACh synthesis was strongly dependent on the presence of Ca++ in the incubation medium. Na+ influx through a TTX-sensitive channel also contributed to this acceleration. Inasmuch as membrane depolarization reduces the initial velocity of Ch uptake and ACh synthesis, their increases during electrical stimulation therefore cannot be the direct effect of the depolarization phase of the action potential. Instead they are the result of the ionic fluxes accompanying the presynaptic spike. It is concluded that stimulation of Ch uptake and ACh synthesis by nerve activity depends first, on the ACh release elicited by Ca++ influx after depolarization and second, on the activation of the Na,K-ATPase due to Na+ entry. Furthermore, it is suggested that the release of ACh after stimulation drives translocation of cytoplasmic ACh into a protected compartment (probably vesicular). This recompartmentation of intraterminal ACh stimulates ACh synthesis by mass action, allowing further accumulation of Ch.     

cGMP-mediated facilitation in nerve terminals by enhancement of the spike afterhyperpolarization

cGMP has long been suspected to play a role in synaptic plasticity, but the inaccessibility of nerve terminals to electrical recording has impeded tests of this hypothesis. In posterior pituitary nerve terminals, nitric oxide enhanced Ca(2+)-activated K+ channel activity by activating guanylate cyclase and PKG. This enhancement occurred only at depolarized potentials, so the spike threshold remained unaltered but the afterhyperpolarization became larger. During spike trains, the enhanced afterhyperpolarization promoted Na+ channel recovery from inactivation, thus reducing action potential failures and allowing more Ca(2+) to enter. Activating guanylate cyclase, either with applied nitric oxide, or with physiological stimulation to activate nitric oxide synthase, increased action potential firing. Thus, the cGMP/nitric oxide cascade generates a short-term, use-dependent enhancement of release.     

Cardioregulatory nerves in the spider Eurypelma marxi Simon

The objective of this study was to locate nerves arising from the CNS that have a cardioregulatory function in the tarantula, Eurypelma marxi Simon. Ramifications of the paired abdominal nerve VIIIb merge with the cardiac ganglion within the first heart segment. Electrical stimulation of the branches of nerve VIIIb that connect with the cardiac ganglion produce changes in heartbeat rate and amplitude. Nerve cutting experiments indicate that no other cardioregulatory nerves are present. Both increases and decreases in heart activity can be produced upon electrical stimulation of nerve VIIIb on each side of the heart. Only one action potential associated with the response of each type could be recorded in each member of the nerve pair. Therefore, we conclude that there are two inhibitory and two acceleratory neurons that arise in the central nervous system to modulate heartbeat activity. The inhibitory effect becomes maximal at a stimulation frequency of 20-30 Hz and the accelerator effect at 30-40 Hz. The aftereffect of acceleratory nerve activity exceeds that of inhibitory nerve activity. When the inhibitor and accelerator are activated simultaneously, the inhibitor dominates. The regulatory nerves interact with neurons in the cardiac ganglion. During inhibition, the number of externally recorded spikes in each ganglionic burst is decreased. The rate and magnitude of the heartbeat are decreased concomitantly. Stimulation of the accelerator enhances electrical activity in the cardiac ganglion at the same time that the heartbeat rate and amplitude are increased.     

Fluorescence Changes in Nerve Induced by Stimulation : Their relation to protein configuration

It was previously assumed, on the basis of changes in the ultraviolet absorption spectrum and of increase in ionizable sulfhydryl groups, that during excitation the proteins of excitable structures undergo some structural rearrangements, and these rearrangements may be similar to those designated by the term transconformation. In the present experiments, it was observed that electrical stimulation of peripheral nerves from rat, guinea pig, frog, and crab causes a decrease in their fluorescence. The peaks of the emission and activation spectra correspond to those attributed to proteins. Denaturing agents, such as urea, were also found to decrease the fluorescence of nerve extracts. It is, therefore, probable that the decrease in fluorescence, associated with the excited state, is due to a change in the configuration of the nerve proteins. The fluorescent method is applicable not only to tissue extracts but allows the observation of surviving nerve fibers before, during, and after stimulation. It showed that fluorescence of the fibers decreases invariably during stimulation and tends to return to the control level during restoration. The reduction in fluorescence is quantitatively related to the number of stimuli received by the nerve.     

Optical detection of neuromodulatory effects of conditioned taste aversion in the pond snail Lymnaea stagnalis

Multiple site optical recording was used to analyze the neural activity changes caused by conditioned taste aversion (CTA) training in the pond snail Lymnaea stagnalis. In response to electrical stimulation of the median lip nerve, which transmits chemosensory signals of appetitive taste to the central nervous system, we optically detected large numbers of spikes in several parts of the buccal ganglion. The effects of CTA training on the spike responses were examined in two areas of the ganglion where the most active neural responses occurred. In one area (termed Area I) that included the N1 medial (N1M) cells, a class of central pattern generator interneurons involved in feeding behavior, the number of spikes in a period 1500-2000 ms after median lip nerve stimulation was significantly reduced in conditioned animals compared to control animals. In another area (termed Area II) positioned between buccal motoneurons, the B3 and B4CL (cluster) cells, the evoked spike responses were unaffected by CTA training. These results, taken together with our previous results indicating an enhancement of an inhibitory input to the N1M cells during CTA, suggest that an appetitive taste signal transmitted to the N1M cells through the median lip nerves is suppressed during CTA, resulting in a decrease of the feeding response.     

Electrophysiological investigation of the cat ciliary ganglion

Electrical responses of some nerves of the ciliary ganglion to stimulation of its other nerves were recorded, and intracellular recordings were also made from neurons of the ganglion (in situ). The overwhelming majority of preganglionic fibers terminate synaptically on neurons of the ganglion. Postganglionic fibers leave in the lateral and medial ciliary nerves, in which the velocity of conduction of excitation ranges from 1.9 to 9.0 m/sec. A few preganglionic fibers pass through the ciliary ganglion into the lateral ciliary nerve, giving off collaterals to neurons of the ganglion, so that stimulation of the lateral ciliary nerve evokes a response in the medial ciliary nerve (preganglionic axon reflex). The resting potential of neurons of the ciliary ganglion is 57±2.8 mV, and their action potential 68±3.6 mV. Single orthodromic stimulation usually evokes a single action potential in a neuron. The amplitude of the EPSP is increased during hyperpolarization of the postsynaptic membrane, confirming the chemical nature of synaptic transmission in the ganglion. The antidromic response consists of an IS-component and spike. The spike is followed by after-hyperpolarization, with a mean amplitude equal to 31% of the spike amplitude, and the time taken for it to fall to one–third of its initial amplitude is 75–135 msec.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 1, pp. 101–108, July–August, 1969.     

Electrical properties of the dendrite in an insect mechanoreceptor: Effects of antidromic or direct electrical stimulation

A study of the negative phase of the spikes recorded extra cellularly from insect mechanoreceptor has been performed in order to characterize some electrical properties of the dendrite which contains the transducing part of the sensory neuron. These properties have been investigated in mechanoreceptors of the metathoracic leg of the locust Schistocerca gregaria by firing antidromic action potentials both at rest and during mechanical or electrical stimulation. The amplitude of the negative phase of the spike appears to be correlated with the polarization of the dendritic membrane, although when bursts of action potentials are applied, the relation is more complex, including a depressive influence of a given spike on the following spike. The receptor potential and the antidromic dendritic spikes both originate in the same region of the dendrite but they involve different ionic processes. Our results indicate that the dendrite is electrically excitable. The spike which originates in the dendrite has an initial negative phase with a small superimposed positive component. A spike of this shape is never observed under natural stimulation. It is proposed that the negative phase of the antidromic impulse provides a suitable means for studying the variations in electrical polarization of the dendrite which cannot be recorded directly.     

Continuous conduction of impulses in peripheral myelinated nerve fibers

1. Conduction of impulses in peripheral myelinated fibers of a nerve trunk is a continuous process, since with uninjured nerve fibers: (a) within each internodal segment the conduction time increases continuously and linearly with increasing conduction distance; (b) the presence of nodes of Ranvier does not result in any detectable discontinuity in the conduction of the impulse; (c) the ascending phase of the spike always has an S shape and never presents signs of fractionation; (d) the shape and magnitude of the spike are constant at all points of each internodal segment. 2. Records have been presented of the external logitudinal current that flows during propagation of an impulse in undissected single nerve fiber (Fig. 6). 3. Propagation of impulses across a conduction block occurs with a readily demonstrable discontinuity.     

The effects of cations upon the action potentials recorded from neurohaemal tissue of the stick insect

Summary The ionic requirement for the action potentials recorded from the neurohaemal tissue on the lateral branch of the median nerve inCarausius morosus has been studied using extracellular electrodes. Sodium-free, magnesium-free, or calcium-free salines produce irreversible block of the action potentials following prolonged exposure to the nerves. Reducing the sodium concentration to 4 mM has little effect on the amplitude of the action potentials, whilst increasing the sodium concentration to 100 mM reduces the amplitude by 50%. Neither tetrodotoxin nor procaine has any effect on these action potentials.Reducing the magnesium concentration to 1 mM increases the amplitude of the action potentials, whilst increasing the concentration of magnesium reduces the amplitude.The amplitude of the action potentials is linearly related to the log of the external calcium concentration, and the action potentials are blocked by both cobalt ions and lanthanum ions.It is concluded that calcium is the major charge carrier of the inward current in these neurosecretory axons which is the first report of calcium dependent action potentials in a nerve axon. Furthermore, small amounts of sodium and magnesium are necessary to maintain electrical activity. Magnesium is a competitive inhibitor of the calcium currents.We are grateful to the Science Research Council for financial support, and to Mrs. J. Birch for the printing of the electron micrographs.     

Membrane Capacitive Memory Alters Spiking in Neurons Described by the Fractional-Order Hodgkin-Huxley Model

Excitable cells and cell membranes are often modeled by the simple yet elegant parallel resistor-capacitor circuit. However, studies have shown that the passive properties of membranes may be more appropriately modeled with a non-ideal capacitor, in which the current-voltage relationship is given by a fractional-order derivative. Fractional-order membrane potential dynamics introduce capacitive memory effects, i.e., dynamics are influenced by a weighted sum of the membrane potential prior history. However, it is not clear to what extent fractional-order dynamics may alter the properties of active excitable cells. In this study, we investigate the spiking properties of the neuronal membrane patch, nerve axon, and neural networks described by the fractional-order Hodgkin-Huxley neuron model. We find that in the membrane patch model, as fractional-order decreases, i.e., a greater influence of membrane potential memory, peak sodium and potassium currents are altered, and spike frequency and amplitude are generally reduced. In the nerve axon, the velocity of spike propagation increases as fractional-order decreases, while in a neural network, electrical activity is more likely to cease for smaller fractional-order. Importantly, we demonstrate that the modulation of the peak ionic currents that occurs for reduced fractional-order alone fails to reproduce many of the key alterations in spiking properties, suggesting that membrane capacitive memory and fractional-order membrane potential dynamics are important and necessary to reproduce neuronal electrical activity.     

Optical Coherence Tomography Phase Measurement of Transient Changes in Squid Giant Axons During Activity

Noncontact optical measurements reveal that transient changes in squid giant axons are associated with action potential propagation and altered under different environmental (i.e., temperature) and physiological (i.e., ionic concentrations) conditions. Using a spectral-domain optical coherence tomography system, which produces real-time cross-sectional images of the axon in a nerve chamber, axonal surfaces along a depth profile are monitored. Differential phase analyses show transient changes around the membrane on a millisecond timescale, and the response is coincident with the arrival of the action potential at the optical measurement area. Cooling the axon slows the electrical and optical responses and increases the magnitude of the transient signals. Increasing the NaCl concentration bathing the axon, whose diameter is decreased in the hypertonic solution, results in significantly larger transient signals during action potential propagation. While monophasic and biphasic behaviors are observed, biphasic behavior dominates the results. The initial phase detected was constant for a single location but alternated for different locations; therefore, these transient signals acquired around the membrane appear to have local characteristics.